DESCRIPTION: This is a Phase I SBIR proposal to develop a simple PCR method to diagnosis alpha-thalassemias. Phase I of the project will conduct feasibility study for the development of a non-isotopic sensitive assay to detect the deletions of five frequently occurring genotypes of alpha- thalassemia using differential PCR. The Investigator propose to use newly developed DNA/PCR mediated color complimentary assay which they developed to test dried blood samples on filter papers. These will be obtained from patients with alpha thalassemic disorders.The specimens will be made available to them by Dr. Griffin Rodgers, the Chief of the Molecular Hematology Unit at the National Institutes of Health. The goal of the project is to develop a diagnostic kit for the detection and quantification of hemoglobin alpha-genes in patients with alpha- thalassemia. It is proposed that the kit will be used for screening potential carriers with alpha- thalassemia in the United States, Southeast Asia and Southern China. This should identify patients at risk of having offspring with symptomatic alpha- thalassemia disorders. The Investigators note correctly that current screening methods are not practical for widespread use and do not differentiate different types of alpha-thalassemia syndromes. In pursuit of the above, they plan to conduct studies on a micro-DNA-PCR assay to detect and differentiate deletions of alpha-hemoglobin genes from dried blood samples. A method for using dried blood samples will be useful in performing field studies. They then plan to establish the feasibility of a DNA-PCR mediated color complementary assay to detect hemoglobin alpha gene deletions from dried blood samples and to combine these two methodologies in to a simple and sensitive diagnostic kit for the alpha-thalassemias. Preliminary work is provided to show the feasibility of the methodology. In these experiments, they demonstrated that deletion of hemoglobin gene alpha1 or alpha2 can be determined using three sets of primers from alpha thalassemia syndrome patients by differential PCR. In preliminary studies, they used this method to examine the hemoglobin alpha gene status from nine normal subjects and seven alpha-thalassemia patients. These studies show that they can clearly differentiate alpha gene deletions in these patients. They can be differentiated by measuring the band intensity of the target chains (alpha1, alpha2) and reference chain (beta-actin) after PCR amplification and gel electrophoresis. They also have applied PCR mediated color complimentary assays on several normal subjects and found this PCR mediated CCA can be used as a rapid sensitive method to detect alpha gene deletions by omitting gel electrophoresis. They postulate that after the assay is improved and stabilized, it would be a useful method of establishing clinical diagnoses in this disorder.